1. Field of the Invention
The present invention relates to isolation and purification of Clostridium botulinum toxins.
2. Description of the Prior Art
Clostridium botulinum strains produce seven immunologically distinct poisonous neurotoxins (NTXs, 7S toxins) and are classified into types A to G. The NTXs inhibit release of acetylcholine at the neuromuscular junctions and synapses, and cause botulism in humans and animals. All types of NTXs are synthesized as a single chain with molecular mass (Mr) of approximately 150 kDa. Endogenous protease(s) from bacteria or exogenous protease (s) such as trypsin cleave the single chain NTXs at about one third of the length from the N-terminus within a region inside a disulfide loop. NTXs therefore become dichain consisting of 50 kDa (designated as light chain) and 100 kDa (designated as heavy chain) components held together by a disulfide bond (FIGS. 1 and 6). The proteolytically processed dichain NTXs (nicked or activated form) are more potent than the single chain NTXs.
The NTXs are associated with nontoxic components in cultures or in foods, and become large complexes designated as progenitor toxins (Oguma et al. , xe2x80x9cStructure and function of Clostridium botulinum progenitor toxin.xe2x80x9d, J. Toxical-Toxin Reviews, 18,17-34, 1999). Type A strain produces three forms of progenitor toxins designated as LL (19S, 900 kDa), L (16S, 500 kDa), and M (12S, 300 kDa) toxins, all of which toxins are considered to be fully activated, while type B, C, and D produce two forms, L and M. Type E, F and G produce only a single form of toxin; types E and F produce M toxin, and type G produces L toxin. M toxin consists of a NTX (7S, 150 kDa) and a nontoxic component showing no hemagglutinin (HA) activity, which is described herein as non-toxic non-HA (NTNH). The molecular mass of NTNH is similar to that of NTX. L and LL toxins are formed by conjugation of M toxin with HA. HA consists of four subcomponents designated as HA1, HA2, HA3a and HA3b (FIG. 6, lanes 2 and 3). NTNH of M toxin is cleaved at its N-terminal regions, and therefore it dissociates into two bands on SDS-PAGE (112 kDa and 15 kDa in FIG. 6, lane 1). M, L and LL toxins dissociate into NTX and nontoxic components under alkaline conditions; M toxin dissociates into NTX and NTNH, and L and LL toxins dissociate into NTX and nontoxic components (NTNH+HA) which are complex of NTNH and HA (FIG. 1).
Recently, partially or almost purified type A-LL toxins and type B-L toxins have been used for the treatment of strabismus, blepharospasm, nystagmus, facial spasm, spasmodic tic, spasmodic torticollis, spastic aphonia, myokymia, bruxism, graphospasm (writer""s cramp), achalasia, anismus, and many other dystonia. The toxins are now used for hidrosis and for elimination of wrinkle, also. Usually, patients are injected with these toxins at several month-intervals, and therefore a significant percentage of the patients produce a specific antibody against the injected toxin. When such anti-toxin antibody is produced in a patient, that toxin type can no longer be used for the same treatment (becomes ineffective) for the patient. It is recommended to use purified fully activated NTXs instead of L and LL toxins in order to reduce antibody production in the patients. Therefore, there has been a demand for a simple procedure to obtain each toxin type in an activated purified form, specially NTX alone.
At present, a type A-LL toxin named xe2x80x9cBOTOXxe2x80x9d that is partially purified from the culture supernatant by repeated precipitation with an acid (pH 5.0 to 3.7) and an alcohol (and ammonium sulfate) (Duff J T et al., xe2x80x9cStudies on immunity to toxins of Clostridium botulinum. A simplified procedure for isolation of type A toxin.xe2x80x9d, J. Bacteriol. 73: 42-47, 1957) is used for the treatment of patients in need thereof. Also, type A-LL toxin and type B-L toxin named xe2x80x9cDYSPORTxe2x80x9d and xe2x80x9cMYOBLOCxe2x80x9d, respectively, have been used. Detailed procedure for purifying these preparations is not clear. However, it is reported that molecular mass of MYOBLOC is 700 kDa and the specific activity of this toxin is relatively low because some of the toxin may be in an un-nicked (un-activated) form. The molecular mass of purified type B-L and -M toxins is reported as 500 and 300 kDa, respectively. Therefore, a 700 kDa preparation seems to be somewhat different from the purified L and M toxins.
The procedure for purifying the type B-L and-M toxins and TX reported by Kozaki, Sakaguchi, and Sakaguchi is as follows: The organisms were incubated in peptone-yeast extract-glucose (PYG) medium at 30xc2x0 C. for 4 days. The precipitate formed by adding 3 N sulfuric acid to pH 4.0 was collected by centrifugation, and the toxins were extracted with 0.2 M phosphate buffer, pH 6.0. The toxins were again precipitated at 20% and then 60% saturation of ammonium sulfate, and the precipitate was dissolved in the same buffer. After treating the extract with ribonuclease, the preparation was adjusted to pH 4.2 with 1 M acetic acid and dialyzed against 0.05 M acetate buffer, pH 4.2. A white precipitate which appeared was collected by centrifugation, washed and then resuspended in 0.05 M acetate buffer, pH 4.2, containing 0.5 M NaCl. This preparation was then treated with protamine (0.06%), and centrifuged. The supernatant was percolated through a sulphopropyl (SP)-Sephadex column equilibrated with 0.05 M acetate buffer containing 0.5 M NaCl to remove the excess protamine. The column effluent was diluted with the 0.05 M acetate buffer to lower the NaCl concentration to 0.2 M, and then applied to a SP-Sephadex column equilibrated with the same buffer with 0.2 M NaCl. The toxin, adsorbed onto the column, was eluted with a linear gradient of NaCl from 0.2 to 0.4 M in the same buffer. The toxic fractions were pooled and concentrated by ultrafiltration, and then applied to a Sephadex G-200 column equilibrated with the same buffer with 0.5 M NaCl. The L and M toxins were obtained separately. From these L and M toxins, NTX was obtained by applying them to a DEAE-Sephadex A-50 column equilibrated with 0.01 M phosphate buffer, pH 8.0. xe2x80x9c(Kozaki S., Sakaguchi S. and Sakaguchi G. Purification and some properties of progenitor toxins of Clostridium botulinum type B. Infect. Immun. 10: 750-756, 1974).
However, conventional purification procedures are very complicated and recovery of toxins is not high. In some strains of types B, C, D and F, fully activated toxins have not been obtained.
An object of the invention is to provide a method for obtaining purified activated toxins in a simple manner.
We have found that hemagglutinin (HA) specifically binds to lactose. Based on this finding, we have developed simple procedures for separating HA-positive and HA-negative progenitor toxins from each other, and also for separating and purifying NTXs from HA-positive progenitor toxins. Furthermore, we have found that if HA-positive progenitor toxins are first treated with trypsin and then applied to a xcex2-lactose column, fully activated HA-positive progenitor toxins that contain little trypsin can be obtained.
Thus, according to the present invention, there is provide a method for separating and purifying HA-positive progenitor toxin(s) (LL and/or L toxins) and HA-negative progenitor toxin (M toxin) from a Clostridium botulinum strain, which comprises applying a liquid containing the HA-positive progenitor toxin(s) and the HA-negative progenitor toxin to a lactose gel column preferably under an acid condition.
There is also provide a method for separating and purifying neurotoxin (7S toxin) from HA-positive progenitor toxins (L and/or LL toxins), which comprises treating the HA-positive progenitor toxins from a Clostridium botulinum strain with an alkaline buffer to dissociate the progenitor toxins into neurotoxin and non-toxic components, and then applying the resulting liquid containing the neurotoxin and the non-toxic components to a lactose gel column.